The penultimate step of the blood coagulation cascade is the Factor Xa-complex-catalyzed conversion of prothrombin to the active enzyme thrombin. Prothrombin is a single-chain, vitamin K-dependent glycoprotein that is synthesized in the liver. It contains a gla domain, two kringle regions, an A chain, and a serine protease domain (B chain). During conversion thrombin, prothrombin is cleaved in two places, removing the gla domain and kringle regions and cleaving between the A and B chains to produce the active protease, α-thrombin. Thrombin is used therapeutically to promote hemostasis in surgery and as a component of tissue adhesives and sealants. Human and bovine thrombins, both derived from plasma, and recombinant human thrombin, are all currently approved for therapeutic use.
Recombinant thrombin is an alternative to plasma-derived thrombin, thus avoiding the potential for contamination that is inherent in plasma-derived products. Ex vivo, active thrombin is produced from prothrombin or variants thereof (e.g., prethrombin-1) by treatment with any of several activating proteases, including those obtained from snake venom. Hence, because of the utility of snake venom proteases in the production of recombinant human thrombin, there is a need for improved recombinant venom-derived proteases that offer, inter alia, higher yield.